Motor proteins and methods for their use

ABSTRACT

The present invention provides high throughput screening systems for identifying compounds useful in the treatment of cellular proliferation disorders. The method can be performed in plurality simultaneously with fluorescence or absorbance readouts.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S.Ser. No. 09/295,612, filed Apr. 20, 1999 pending and of U.S. Ser. No.09/314,464, filed May 18, 1999 pending each of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to methods for the identification of compoundsthat modulate the activity of target proteins having motor domains anduse of such methods for the identification of therapeutic agents.

BACKGROUND OF THE INVENTION

The kinesin superfamily is an extended family of related microtubulemotor proteins. It can be classified into at least 8 subfamilies basedon primary amino acid sequence, domain structure, velocity of movement,and cellular function. This family is exemplified by “true” kinesin,which was first isolated from the axoplasm of squid, where it isbelieved to play a role in anterograde axonal transport of vesicles andorganelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)).

Mitotic kinesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures. Mitotic kinesins play essential roles during all phases ofmitosis. These enzymes are “molecular motors” that translate energyreleased by hydrolysis of ATP into mechanical force which drives thedirectional movement of cellular cargoes along microtubules. Thecatalytic domain sufficient for this task is a compact structure ofapproximately 340 amino acids. During mitosis, kinesins organizemicrotubules into the bipolar spindle that is the mitotic spindle.Kinesins mediate movement of chromosomes along spindle microtubules, aswell as structural changes in the mitotic spindle associated withspecific phases of mitosis. Experimental perturbation of mitotic kinesinfunction causes malformation or dysfunction of the mitotic spindle,frequently resulting in cell cycle arrest.

Within this functional group of kinesins resides a group of kinesinsfrom several organisms that share significant sequence homology, the KinI subfamily, and that function to destabilize microtubule ends. Theseinclude H. sapiens MCAK (also known as mitotic centromere-associatedkinesin or HsMCAK), X laevis MCAK, and C. griseus MCAK.

During anaphase A, disjoined sister chromatids migrate poleward. Thispoleward movement is driven by kinetochores and is accompanied by thedepolymerization of microtubules attached to the migrating chromatids.The kinesin MCAK plays an important role in this motility and maypromote disassembly of microtubules attached to kinetochores of mitoticchromosomes.

The HsMCAK gene has a predicted 723 amino acid open reading frame,encoding a 81 kDa protein that shares 79.2% homology with hamster MCAK.HsMCAK is expressed in tissues containing dividing cells, such asthymus, testis, small intestine, colon (mucosal lining), and placenta.Genes for the Xenopus and hamster homologs of MCAK has also been clonedand characterized.

Defects in function of these proteins would be expected to result incell cycle arrest in mitosis. As such, compounds that modulate theactivity of these kinesins may affect cellular proliferation. Thepresent invention provides a novel method to identify such compounds.

SUMMARY OF THE INVENTION

The present invention provides methods to identify candidate agents thatbind to a target protein or act as a modulator of the bindingcharacteristics or biological activity of a target protein. In oneembodiment, the method is performed in plurality simultaneously. Forexample, the method can be performed at the same time on multiple assaymixtures in a multi-well screening plate. Furthermore, in a preferredembodiment, fluorescence or absorbance readouts are utilized todetermine activity. Thus, in one aspect, the invention provides a highthroughput screening system for detecting modulators of activity atarget protein.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate, under conditions that normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to areaction that uses said ADP or phosphate as a substrate under conditionsthat normally allow the ADP or phosphate to be utilized and determiningthe level of activity of the reaction as a measure of the concentrationof ADP or phosphate. A change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

The phrase “use ADP or phosphate” means that the ADP or phosphate aredirectly acted upon by detection reagents. In one case, the ADP, forexample, can be hydrolyzed or can be phosphorylated. As another example,the phosphate can be added to another compound. As used herein, in eachof these cases, ADP or phosphate is acting as a substrate.

Preferably, the target protein either directly or indirectly producesADP or phosphate and comprises a motor domain. More preferably, thetarget protein comprises HsMCAK, X laevis MCAK, and C. griseus MCAK or afragment thereof. Most preferably, the target protein comprises SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,or SEQ ID NO:14.

Also provided are modulators of the target protein including agents forthe treatment of cellular proliferation, including cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation. Theagents and compositions provided herein can be used in variety ofapplications which include the formulation of sprays, powders, and othercompositions. Also provided herein are methods of treating cellularproliferation disorders such as cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation, for treatingdisorders associated with MCAK activity, and for inhibiting MCAK.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:1).

FIG. 2 shows an embodiment of a particularly preferred target protein(SEQ ID NO:2). The construct contains residues 189 through 617 of thefull length MCAK enzyme.

FIG. 3 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:3).

FIG. 4 shows an embodiment of another particularly preferred targetprotein (SEQ ID NO:4). The construct contains residues 228 through 617of the full length MCAK enzyme.

FIG. 5 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:5).

FIG. 6 shows an embodiment of another particularly preferred targetprotein (SEQ ID NO:6). The construct contains residues 257 through 617of the full length MCAK enzyme.

FIG. 7 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:7).

FIG. 8 shows an embodiment of a particularly preferred target protein(SEQ ID NO:8). The construct contains residues 189 through 660 of thefull length MCAK enzyme.

FIG. 9 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:9).

FIG. 10 shows an embodiment of a particularly preferred target protein(SEQ ID NO:10). The construct contains residues 228 through 660 of thefull length MCAK enzyme.

FIG. 11 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:11).

FIG. 12 shows an embodiment of a particularly preferred target protein(SEQ ID NO:12). The construct contains residues 257 through 660 of thefull length MCAK enzyme.

FIG. 13 shows an embodiment of a nucleic acid sequence encoding aparticularly preferred target protein (SEQ ID NO:13).

FIG. 14 shows an embodiment of a particularly preferred target protein(SEQ ID NO:14). The construct contains residues 3 through 725 of thefull length MCAK enzyme.

FIGS. 15A and 15B show a nucleic acid sequence encoding HsMCAK (SEQ IDNO:15).

FIG. 16 shows the amino acid sequence of HsMCAK (SEQ ID NO:16).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“ADP” refers to adenosine diphosphate and also includes ADP analogs,including, but not limited to, deoxyadenosine diphosphate (dADP) andadenosine analogs.

“Biologically active” target protein refers to a target protein that hasone or more of kinesin protein's biological activities, including, butnot limited to microtubule stimulated ATPase activity, as tested, e.g.,in an ATPase assay. Biological activity can also be demonstrated in amicrotubule gliding assay or a microtubule binding assay. “ATPaseactivity” refers to ability to hydrolyze ATP. Other activities includepolymerization/depolymerization (effects on microtubule dynamics),binding to other proteins of the spindle, binding to proteins involvedin cell-cycle control, or serving as a substrate to other enzymes, suchas kinases or proteases and specific kinesin cellular activities, suchas chromosome congregation, axonal transport, etc.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a target protein or a fragment thereof or nucleicacid encoding a target protein or a fragment thereof. Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. A biological sample comprises at least one cell,preferably plant or vertebrate. Embodiments include cells obtained froma eukaryotic organism, preferably eukaryotes such as fungi, plants,insects, protozoa, birds, fish, reptiles, and preferably a mammal suchas rat, mice, cow, dog, guinea pig, or rabbit, and most preferably aprimate such as chimpanzees or humans.

A “comparison window’ includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a dendrogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). As ageneral rule, PileUp can align up to 500 sequences, with any singlesequence in the final alignment restricted to a maximum length of 7,000characters.

The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster can then be aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences canbe aligned by a simple extension of the pairwise alignment of twoindividual sequences. A series of such pairwise alignments that includesincreasingly dissimilar sequences and clusters of sequences at eachiteration produces the final alignment.

“Variant” applies to both amino acid and nucleic acid sequences. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

Also included within the definition of target proteins of the presentinvention are amino acid sequence variants of wild-type target proteins.These variants fall into one or more of three classes: substitutional,insertional or deletional variants. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe target protein, using cassette or PCR mutagenesis or othertechniques well known in the art, to produce DNA encoding the variant,and thereafter expressing the DNA in recombinant cell culture. Varianttarget protein fragments having up to about 100-150 amino acid residuesmay be prepared by in vitro synthesis using established techniques.Amino acid sequence variants are characterized by the predeterminednature of the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variation of the target protein aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally occurring analogue, althoughvariants can also be selected which have modified characteristics.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to about 20 amino acids,although considerably longer insertions may be tolerated. Deletionsrange from about 1 to about 20 residues, although in some cases,deletions may be much longer.

Substitutions, deletions, and insertions or any combinations thereof maybe used to arrive at a final derivative. Generally, these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger characteristics may be tolerated in certaincircumstances.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).

“Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. Includes cytoskeletal polymers (e.g., actin filaments,microtubules, intermediate filaments, myosin fragments), molecularmotors (e.g., kinesins, myosins, dyneins), cytoskeleton associatedregulatory proteins (e.g., tropomysin, alpha-actinin) and cytoskeletalassociated binding proteins (e.g., microtubules associated proteins,actin binding proteins).

“Cytoskeletal function” refers to biological roles of the cytoskeleton,including but not limited to the providing of structural organization(e.g., microvilli, mitotic spindle) and the mediation of motile eventswithin the cell (e.g., muscle contraction, mitotic chromosome movements,contractile ring formation and function, pseudopodal movement, activecell surface deformations, vesicle formation and translocation.)

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

“High stringency conditions” may be identified by those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 jig/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride / sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

“High throughput screening” as used herein refers to an assay whichprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, or plant cells. Both primary cells and cultured cell lines areincluded in this definition.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is 50or 100 amino acids in length. This definition also refers to thecomplement of a test sequence, provided that the test sequence has adesignated or substantial identity to a reference sequence. Preferably,the percent identity exists over a region of the sequence that is atleast about 25 nucleotides in length, more preferably over a region thatis 50 or 100 nucleotides in length.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g,. charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. The scoring of conservative substitutions can becalculated according to, e.g., the algorithm of Meyers & Millers,Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, Calif.).

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated target gene, the nucleic acid of interest is separated fromopen reading frames which flank the target gene and encode proteinsother than the target protein. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include fluorescent proteins such as green, yellow, red or bluefluorescent proteins, radioisotopes such as ³²p, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g., the polypeptide of SEQ IDNO:2 can be made detectable, e.g., by incorporating a radio-label intothe peptide, and used to detect antibodies specifically reactive withthe peptide).

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μ/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

“Modulators,” “inhibitors,” and “activators of a target protein” referto modulatory molecules identified using in vitro and in vivo assays fortarget protein activity. Such assays include ATPase activity,microtubule gliding, microtubule depolymerizing activity, and bindingactivity such as microtubule binding activity or binding of nucleotideanalogs. Samples or assays that are treated with a candidate agent at atest and control concentration. The control concentration can be zero.If there is a change in target protein activity between the twoconcentrations, this change indicates the identification of a modulator.A change in activity, which can be an increase or decrease, ispreferably a change of at least 20% to 50%, more preferably by at least50% to 75%, more preferably at least 75% to 100%, and more preferably150% to 200%, and most preferably is a change of at least 2 to 10 foldcompared to a control. Additionally, a change can be indicated by achange in binding specificity or substrate.

“Molecular motor” refers to a molecule that utilizes chemical energy togenerate mechanical force. According to one embodiment, the molecularmotor drives the motile properties of the cytoskeleton.

The phrase “motor domain” refers to the domain of a target protein thatconfers membership in the kinesin superfamily of motor proteins througha sequence identity of approximately 35-45% identity to the motor domainof true kinesin.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in eiter single- or double-strandedform. Unless specifically limited, the term encompasses nucleic acidscontaining known analogues of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. For example, degenerate codon substitutions may beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260)2605-2608 (1985); Cassol et al. 1992;Rossolini et al. Mol. Cell. Probes 8:91-98 (1994)). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

“Nucleic acid probe or oligonucleotide” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, or T) ormodified bases. In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidine complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. A target protein comprises a polypeptide demonstrated to haveat least microtubule stimulated ATPase activity. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by Nomenclature Commission. Nucleotides, likewise, may be referred toby their commonly accepted single-letter codes, i.e., the one-lettersymbols recommended by the IUPAC-IUB. A “promoter” is defined as anarray of nucleic acid control sequences that direct transcription of anucleic acid. As used herein, a promoter includes necessary nucleic acidsequences near the start site of transcription, such as, in the case ofa polymerase II type promoter, a TATA box element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. A “constitutive” promoter is a promoter that is activeunder most environmental and developmental conditions. An “inducible”promoter is a promoter that is under environmental or developmentalregulation. The term “operably linked” refers to a functional linkagebetween a nucleic acid expression control sequence (such as a promoter,or array of transcription factor binding sites) and a second nucleicacid sequence, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised tothe target protein with the amino acid sequence encoded in SEQ ID NO:2can be selected to obtain only those antibodies that are specificallyimmunoreactive with the target protein and not with other proteins,except for polymorphic variants, orthologs, alleles, and closely relatedhomologues of MCAK. This selection may be achieved by subtracting outantibodies that cross react with molecules, for example, such as C.elegans unc-104 and human Kif1A. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the cytoskeletal system in vivo which can haveapplication in both human and animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding, but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders such as rheumatoidarthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic disesase such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma, andOsler Webber syndrome.

II. The Target Protein

According to the present invention, a target protein is a molecule thateither directly or indirectly produces ADP or phosphate and thatcomprises a motor domain. In a preferred embodiment, the target proteinis an enzyme having activity which produces ADP and/or phosphate as areaction product. Also included within the definition of the targetproteins are amino acid sequence variants of wild-type target proteins.

Target proteins of the present invention may also be modified in a wayto form chimeric molecules comprising a fusion of a target protein witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affmity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al., (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD)) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Hopp etal. (1988) BioTechnology 6:1204); the KT3 epitope peptide (see, Martineet al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner(1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag(see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.Target proteins of the present invention are meant to include both theuntagged target protein as well as the chimeric protein wherein thetarget protein has been fused to one or more tag epitopes.

In a particularly preferred embodiment, the target protein comprisesHsMCAK, X laevis MCAK, and C. griseus MCAK or a fragment thereof.

In another aspect of this invention, the target protein comprises anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, or SEQ ID NO:14, preferably greater than 80%, more preferablygreater than 90%, more preferably greater than 95% or, in anotherembodiment, has 98 to 100% sequence identity with SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ IDNO:14.

In a particularly preferred embodiment, a fragment of the HsMCAK proteincomprising a portion of its hydrolytically active “motor” domain isused. This motor domain has been cloned and expressed in bacteria suchthat large quantities of biochemically active, substantially pureprotein are available. Preferably, the target protein comprises an aminoacid sequence which has greater than 70% sequence identity with SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,or SEQ ID NO:14, preferably greater than 80%, more preferably greaterthan 90%, more preferably greater than 95% or, in another embodiment,has 98 to 100% sequence identity with SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.

A particularly preferred embodiment is drawn to a fragment of the HsMCAKprotein SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, or SEQ ID NO:14. More preferably, this fragment istagged at the C-terminus with a myc epitope and 6 histidines. Morepreferably, this fragment is tagged at the N-terminus with a T7 epitopeand at the C-terminus with a myc epitope and 6 histidines.

In one aspect, the nucleic acids provided herein are defined by theproteins encoded thereby. A preferred embodiment of the invention isdrawn to an isolated nucleic acid sequence encoding a microtubule motorprotein, wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 70% sequenceidentity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, or SEQ ID NO:14, preferably greater than 80%, morepreferably greater than 90%, more preferably greater than 95% or, inanother embodiment, has 98 to 100% sequence identity with SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, orSEQ ID NO:14. In one embodiment, the nucleic acid encodes HsMCAK or afragment thereof. In another embodiment, the nucleic acid encodes SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,or SEQ ID NO:14.

In one embodiment, the nucleic acid comprises a sequence which has oneor more of the following characteristics: greater than 55 or 60%sequence identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13,preferably greater than 70%, more preferably greater than 80%, morepreferably greater than 90 or 95% or, in another embodiment, has 98 to100% sequence identity with SEQID NO:1, SEQIDNO:3, SEQID NO:5,SEQIDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13. Inanother embodiment provided herein, the nucleic acid hybridizes understringent conditions to a nucleic acid having a sequence orcomplementary sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13. Inanother embodiment, the nucleic acid has a nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:11, or SEQ ID NO:13. As described above, when describing thenucleotide in terms of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:13, thesequence identity may be slightly lower due to the degeneracy in thegenetic code.

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the protein.

Numerous suitable methods for recombinant protein expression, includinggeneration of expression vectors, generation of fusion proteins,introducing expression vectors into host cells, protein expression inhost cells, and purifications methods are known to those in the art.

In a preferred embodiment, the target proteins are purified for use inthe assays to provide substantially pure samples. Alternatively, thetarget protein need not be substantially pure as long as the samplecomprising the target protein is substantially free of other componentsthat can contribute to the production of ADP or phosphate.

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocussing. For example,the target protein can be purified using a standard anti-target antibodycolumn. Ultrafiltration and diafiltration techniques, in conjunctionwith protein concentration, are also useful.

Either naturally occurring or recombinant target protein can be purifiedfor use in functional assays. The target protein may be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others (see, e.g.,Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra). Apreferred method of purification is use of Ni-NTA agarose (Qiagen).

Suitable purification schemes for some specific kinesins are outlined inU.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby expresslyincorporated herein in its entirety for all purposes.

The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays provided herein such as those which assay ATPaseactivity or microtubule binding activity. Biologically active targetprotein is useful for identifying modulators of target protein orfragments thereof and kinesin superfamily members using in vitro assayssuch as microtubule gliding assays, ATPase assays (Kodama et al., J.Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA90:5209-5213 (1993)), and binding assays including microtubule bindingassays (Vale et al., Cell 42:39-50 (1985)), as described in detailbelow.

III. Assays for modulators of the target protein

A. Functional assays

Assays that can be used to test for modulators of the target proteininclude a variety of in vitro or in vivo assays, e.g., microtubulegliding assays, binding assays such as microtubule binding assays,microtubule depolymerization assays, and ATPase assays (Kodama et al.,J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci.USA 90: 5209-5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115(1995); (Vale et al., Cell 42:39-50 (1985)).

Modulation is tested by screening for candidate agents capable ofmodulating the activity of the target protein comprising the steps ofcombining a candidate agent with the target protein, as above, anddetennining an alteration in the biological activity of the targetprotein. Thus, in this embodiment, the candidate agent should both bindto the target protein (although this may not be necessary), and alterits biological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods and in vivo screening of cellsfor alterations in cell cycle distribution, cell viability, or for thepresence, morphology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

In a preferred embodiment, molecular motor activity is measured by themethods disclosed in Ser. No. 09/314,464, filed May 18, 1999, entitled“Compositions and assay utilizing ADP or phosphate for detecting proteinmodulators”, which is incorporated herein by reference in its entirety.More specifically, this assay detects modulators of any aspect of akinesin motor function ranging from interaction with microtubules tohydrolysis of ATP. ADP or phosphate is used as the readout for proteinactivity.

There are a number of enzymatic assays known in the art which use ADP asa substrate. For example, kinase reactions such as pyruvate kinases areknown. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31 (1970). Thisis a preferred method in that it allows the regeneration of ATP. In oneembodiment, the level of activity of the enzymatic reaction isdetermined directly. In a preferred embodiment, the level of activity ofthe enzymatic reaction which uses ADP as a substrate is measuredindirectly by being coupled to another reaction. For example, in oneembodiment, the method further comprises a lactate dehydrogenasereaction under conditions which normally allow the oxidation of NADH,wherein said lactate dehydrogenase reaction is dependent on the pyruvatekinase reaction. Measurement of enzymatic reactions by coupling is knownin the art. Furthermore, there are a number of reactions which utilizephosphate. Examples of such reactions include a purine nucleosidephosphorylase reaction. This reaction can be measured directly orindirectly. A particularly preferred embodiments utilizes the pyruvatekinase/lactate dehydrogenase system.

In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example, by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. One method of quantifying the phosphomolybdateis with malachite green. Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

In addition, target protein activity can be examined by determiningmodulation of target protein in vitro using cultured cells. The cellsare treated with a candidate agent and the effect of such agent on thecells is then determined either directly or by examining relevantsurrogate markers. For example, characteristics such as mitotic spindlemorphology and cell cycle distribution can be used to determine theeffect.

Thus, in a preferred embodiment, the methods comprise combining a targetprotein and a candidate agent, and determining the effect of thecandidate agent on the target protein. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

As will be appreciated by those in the art, the components may be addedin buffers and reagents to assay target protein activity and giveoptimal signals. Since the methods allow kinetic measurements, theincubation periods can be optimized to give adequate detection signalsover the background.

In a preferred embodiment, an antifoam or a surfactant is included inthe assay mixture. Suitable antifoams include, but are not limited to,antifoam 289 (Sigma). Suitable surfactants include, but are not limitedto, Tween, Tritons, including Triton X-100, saponins, andpolyoxyethylene ethers. Generally, the antifoams, detergents, orsurfactants are added at a range from about 0.01 ppm to about 10 ppm.

A preferred assay design is also provided. In one aspect, the inventionprovides a multi-time-point (kinetic) assay, with at least two datapoints being preferred. In the case of multiple measurements, theabsolute rate of the protein activity can be deterinined.

B. Binding Assays

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget protein, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the candidate agent and the binding moiety, with the bindingmoiety displacing the candidate agent.

Competitive screening assays may be done by combining the target proteinand a drug candidate in a first sample. A second sample comprises acandidate agent, the target protein and a compound that is known tomodulate the target protein. This may be performed in either thepresence or absence of microtubules. The binding of the candidate agentis determined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the target protein and potentially modulating its activity.That is, if the binding of the candidate agent is different in thesecond sample relative to the first sample, the candidate agent iscapable of binding to the target protein.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to the target proteinfor a time sufficient to allow binding. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to the target protein and thus is capable ofbinding to, and potentially modulating, the activity of the targetprotein. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the washsolution indicates displacement by the agent. Alternatively, if thecandidate agent is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound tothe target protein with a higher affinity. Thus, if the candidate agentis labeled, the presence of the label on the support, coupled with alack of competitor binding, may indicate the candidate agent is capableof binding to the target protein.

C. Candidate agents

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. In a preferred embodiment,the candidate agents are organic chemical moieties, a wide variety ofwhich are available in the literature.

D. Other assay components

The assays provided utilize target protein as defined herein. In oneembodiment, portions of target protein are utilized; in a preferredembodiment, portions having target protein activity as described hereinare used. In addition, the assays described herein may utilize eitherisolated target proteins or cells or animal models comprising the targetproteins.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Also,reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

IV. Applications

The methods of the invention are used to identify compounds useful inthe treatment of cellular proliferation diseases. Disease states whichcan be treated by the methods and compositions provided herein include,but are not limited to, cancer (further discussed below), autoimmunedisease, arthritis, graft rejection, inflammatory bowel disease,proliferation induced after medical procedures, including, but notlimited to, surgery, angioplasty, and the like. It is appreciated thatin some cases the cells may not be in a hyper or hypo proliferationstate (abnormal state) and still require treatment. For example, duringwound healing, the cells may be proliferating “normally”, butproliferation enhancement may be desired. Similarly, as discussed above,in the agriculture arena, cells may be in a “normal” state, butproliferation modulation may be desired to enhance a crop by directlyenhancing growth of a crop, or by inhibiting the growth of a plant ororganism which adversely affects the crop. Thus, in one embodiment, theinvention herein includes application to cells or individuals afflictedor impending affliction with any one of these disorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesotheliorna;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); G necological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma],fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above identified conditions.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant administration of atherapeutically effective dose of the candidate agents of the inventionto a cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Candidate agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt.%. The agents maybe administered alone or incombination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifiing agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations. The administration of the candidate agentsof the present invention can be done in a variety of ways as discussedabove, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.In some instances, for example, in the treatment of wounds andinflammation, the candidate agents may be directly applied as a solutionor spray.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein. Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for ATPase assays, microtubule gliding assays, or microtubulebinding assays.

V. Examples

This assay is based on detection of ADP production from a targetprotein's microtubule stimulated ATPase. ATPproduction is monitored by acoupled enzyme system consisting of pyruvate kinase and lactatedehydrogenase. Under the assay conditions described below, pyruvatekianse catalyzes the conversion of ADP and phosphoenol pyruvate topyruvate and ATP. Lactate dehydrogenase then catalyzes theoxidation-reduction reaction of pyruvate and NADH to lactate and NAD+.Thus, for each molecule of ADP produced, one molecule of NADH isconsumed. The amount of NADH in the assay solution is monitored bymeasuring light absorbance at a wavelength of 340 nm.

The final 25 μl assay solution consists of the following: 5 μg/ml targetprotein, 30 μg/ml nicrotubules, 5 μM Taxol, 0.8 mM NADH, 1.5 mMphosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactatedehydrogenase, 25 mM Pipes/KOH pH 6.8, 2mM MgCl₂, 1 mM EGTA, 1 mM MDTT,0.1 mg/ml BSA, 0.001% antifoam 289, and 1 mM ATP.

Potential candidate agents are dissolved in DMSO at a concentration ofabout 1 mg/ml and 0.5 μl of each chemical solution is dispensed into asingle well of a clear 384 well plate. Each of the 384 wells are thenfilled with 20 μl of a solution consisting of all of the assaycomponents described above except for ATP. The plate is agitated at ahigh frequency. To start the assay, 5 μl of a solution containing ATP isadded to each well. The plate is agitated and the absorbance is read at340 nm over various time intervals. The assay is run at roomtemperature.

The assay components and the performance of the assay are optimizedtogether to match the overall read time with the rate of the targetprotein's ADP production. The read time should be long enough for therate of NADH consumption to reach steady state beyond an initial lagtime of several seconds.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

16 1 1292 DNA Human 1 atgggaggaa atcatgtctt gtgaaggaag tggaaaaaatgaagaacaag cgagaagaga 60 agaaggccca gaactctgaa atgagaatga agagagctcaggagtatgac agtagttttc 120 caaactggga atttgcccga atgattaaag aatttcgggctactttggaa tgtcatccac 180 ttactatgac tgatcctatc gaagagcaca gaatatgtgtctgtgttagg aaacgcccac 240 tgaataagca agaattggcc aagaaagaaa ttgatgtgatttccattcct agcaagtgtc 300 tcctcttggt acatgaaccc aagttgaaag tggacttaacaaagtatctg gagaaccaag 360 cattctgctt tgactttgca tttgatgaaa cagcttcgaatgaagttgtc tacaggttca 420 cagcaaggcc actggtacag acaatctttg aaggtggaaaagcaacttgt tttgcatatg 480 gccagacagg aagtggcaag acacatacta tgggcggagacctctctggg aaagcccaga 540 atgcatccaa agggatctat gccatggcct cccgggacgtcttcctcctg aagaatcaac 600 cctgctaccg gaagttgggc ctggaagtct atgtgacattcttcgagatc tacaatggga 660 agctgtttga cctgctcaac aagaaggcca agctgcgcgtgctggaggat ggcaagcaac 720 aggtgcaagt ggtggggctg caggagcatc tggttaactctgctgatgat gtcatcaaga 780 tgatcgacat gggcagcgcc tgcagaacct ctgggcagacatttgccaac tccaattcct 840 cccgctccca cgcgtgcttc caaattattc ttcgagctaaagggagaatg catggcaagt 900 tctctttggt agatctggca gggaatgagc gaggcgcggacacttccagt gctgaccggc 960 agacccgcat ggagggcgca gaaatcaaca agagtctcttagccctgaag gagtgcatca 1020 gggccctggg acagaacaag gctcacaccc cgttccgtgagagcaagctg acacaggtgc 1080 tgagggactc cttcattggg gagaactcta ggacttgcatgattgccacg atctcaccag 1140 gcataagctc ctgtgaatat actttaaaca ccctgagatatgcagacagg gtcaaggagc 1200 tgagccccca cagtgggccc agtggagagc agttgattcaaatggaaaca gaagagatgg 1260 aagcctgctc taacggggcg ctgattccat ga 1292 2430 PRT Human 2 Met Arg Arg Lys Ser Cys Leu Val Lys Glu Val Glu Lys MetLys Asn 1 5 10 15 Lys Arg Glu Glu Lys Lys Ala Gln Asn Ser Glu Met ArgMet Lys Arg 20 25 30 Ala Gln Glu Tyr Asp Ser Ser Phe Pro Asn Trp Glu PheAla Arg Met 35 40 45 Ile Lys Glu Phe Arg Ala Thr Leu Glu Cys His Pro LeuThr Met Thr 50 55 60 Asp Pro Ile Glu Glu His Arg Ile Cys Val Cys Val ArgLys Arg Pro 65 70 75 80 Leu Asn Lys Gln Glu Leu Ala Lys Lys Glu Ile AspVal Ile Ser Ile 85 90 95 Pro Ser Lys Cys Leu Leu Leu Val His Glu Pro LysLeu Lys Val Asp 100 105 110 Leu Thr Lys Tyr Leu Glu Asn Gln Ala Phe CysPhe Asp Phe Ala Phe 115 120 125 Asp Glu Thr Ala Ser Asn Glu Val Val TyrArg Phe Thr Ala Arg Pro 130 135 140 Leu Val Gln Thr Ile Phe Glu Gly GlyLys Ala Thr Cys Phe Ala Tyr 145 150 155 160 Gly Gln Thr Gly Ser Gly LysThr His Thr Met Gly Gly Asp Leu Ser 165 170 175 Gly Lys Ala Gln Asn AlaSer Lys Gly Ile Tyr Ala Met Ala Ser Arg 180 185 190 Asp Val Phe Leu LeuLys Asn Gln Pro Cys Tyr Arg Lys Leu Gly Leu 195 200 205 Glu Val Tyr ValThr Phe Phe Glu Ile Tyr Asn Gly Lys Leu Phe Asp 210 215 220 Leu Leu AsnLys Lys Ala Lys Leu Arg Val Leu Glu Asp Gly Lys Gln 225 230 235 240 GlnVal Gln Val Val Gly Leu Gln Glu His Leu Val Asn Ser Ala Asp 245 250 255Asp Val Ile Lys Met Ile Asp Met Gly Ser Ala Cys Arg Thr Ser Gly 260 265270 Gln Thr Phe Ala Asn Ser Asn Ser Ser Arg Ser His Ala Cys Phe Gln 275280 285 Ile Ile Leu Arg Ala Lys Gly Arg Met His Gly Lys Phe Ser Leu Val290 295 300 Asp Leu Ala Gly Asn Glu Arg Gly Ala Asp Thr Ser Ser Ala AspArg 305 310 315 320 Gln Thr Arg Met Glu Gly Ala Glu Ile Asn Lys Ser LeuLeu Ala Leu 325 330 335 Lys Glu Cys Ile Arg Ala Leu Gly Gln Asn Lys AlaHis Thr Pro Phe 340 345 350 Arg Glu Ser Lys Leu Thr Gln Val Leu Arg AspSer Phe Ile Gly Glu 355 360 365 Asn Ser Arg Thr Cys Met Ile Ala Thr IleSer Pro Gly Ile Ser Ser 370 375 380 Cys Glu Tyr Thr Leu Asn Thr Leu ArgTyr Ala Asp Arg Val Lys Glu 385 390 395 400 Leu Ser Pro His Ser Gly ProSer Gly Glu Gln Leu Ile Gln Met Glu 405 410 415 Thr Glu Glu Met Glu AlaCys Ser Asn Gly Ala Leu Ile Pro 420 425 430 3 1175 DNA Human 3atgcaaactg ggaatttgcc cgaatgatta aagaatttcg ggctactttg gaatgtcatc 60cacttactat gactgatcct atcgaagagc acagaatatg tgtctgtgtt aggaaacgcc 120cactgaataa gcaagaattg gccaagaaag aaattgatgt gatttccatt cctagcaagt 180gtctcctctt ggtacatgaa cccaagttga aagtggactt aacaaagtat ctggagaacc 240aagcattctg ctttgacttt gcatttgatg aaacagcttc gaatgaagtt gtctacaggt 300tcacagcaag gccactggta cagacaatct ttgaaggtgg aaaagcaact tgttttgcat 360atggccagac aggaagtggc aagacacata ctatgggcgg agacctctct gggaaagccc 420agaatgcatc caaagggatc tatgccatgg cctcccggga cgtcttcctc ctgaagaatc 480aaccctgcta ccggaagttg ggcctggaag tctatgtgac attcttcgag atctacaatg 540ggaagctgtt tgacctgctc aacaagaagg ccaagctgcg cgtgctggag gatggcaagc 600aacaggtgca agtggtgggg ctgcaggagc atctggttaa ctctgctgat gatgtcatca 660agatgatcga catgggcagc gcctgcagaa cctctgggca gacatttgcc aactccaatt 720cctcccgctc ccacgcgtgc ttccaaatta ttcttcgagc taaagggaga atgcatggca 780agttctcttt ggtagatctg gcagggaatg agcgaggcgc ggacacttcc agtgctgacc 840ggcagacccg catggagggc gcagaaatca acaagagtct cttagccctg aaggagtgca 900tcagggccct gggacagaac aaggctcaca ccccgttccg tgagagcaag ctgacacagg 960tgctgaggga ctccttcatt ggggagaact ctaggacttg catgattgcc acgatctcac 1020caggcataag ctcctgtgaa tatactttaa acaccctgag atatgcagac agggtcaagg 1080agctgagccc ccacagtggg cccagtggag agcagttgat tcaaatggaa acagaagaga 1140tggaagcctg ctctaacggg gcgctgattc catga 1175 4 391 PRT Human 4 Met ProAsn Trp Glu Phe Ala Arg Met Ile Lys Glu Phe Arg Ala Thr 1 5 10 15 LeuGlu Cys His Pro Leu Thr Met Thr Asp Pro Ile Glu Glu His Arg 20 25 30 IleCys Val Cys Val Arg Lys Arg Pro Leu Asn Lys Gln Glu Leu Ala 35 40 45 LysLys Glu Ile Asp Val Ile Ser Ile Pro Ser Lys Cys Leu Leu Leu 50 55 60 ValHis Glu Pro Lys Leu Lys Val Asp Leu Thr Lys Tyr Leu Glu Asn 65 70 75 80Gln Ala Phe Cys Phe Asp Phe Ala Phe Asp Glu Thr Ala Ser Asn Glu 85 90 95Val Val Tyr Arg Phe Thr Ala Arg Pro Leu Val Gln Thr Ile Phe Glu 100 105110 Gly Gly Lys Ala Thr Cys Phe Ala Tyr Gly Gln Thr Gly Ser Gly Lys 115120 125 Thr His Thr Met Gly Gly Asp Leu Ser Gly Lys Ala Gln Asn Ala Ser130 135 140 Lys Gly Ile Tyr Ala Met Ala Ser Arg Asp Val Phe Leu Leu LysAsn 145 150 155 160 Gln Pro Cys Tyr Arg Lys Leu Gly Leu Glu Val Tyr ValThr Phe Phe 165 170 175 Glu Ile Tyr Asn Gly Lys Leu Phe Asp Leu Leu AsnLys Lys Ala Lys 180 185 190 Leu Arg Val Leu Glu Asp Gly Lys Gln Gln ValGln Val Val Gly Leu 195 200 205 Gln Glu His Leu Val Asn Ser Ala Asp AspVal Ile Lys Met Ile Asp 210 215 220 Met Gly Ser Ala Cys Arg Thr Ser GlyGln Thr Phe Ala Asn Ser Asn 225 230 235 240 Ser Ser Arg Ser His Ala CysPhe Gln Ile Ile Leu Arg Ala Lys Gly 245 250 255 Arg Met His Gly Lys PheSer Leu Val Asp Leu Ala Gly Asn Glu Arg 260 265 270 Gly Ala Asp Thr SerSer Ala Asp Arg Gln Thr Arg Met Glu Gly Ala 275 280 285 Glu Ile Asn LysSer Leu Leu Ala Leu Lys Glu Cys Ile Arg Ala Leu 290 295 300 Gly Gln AsnLys Ala His Thr Pro Phe Arg Glu Ser Lys Leu Thr Gln 305 310 315 320 ValLeu Arg Asp Ser Phe Ile Gly Glu Asn Ser Arg Thr Cys Met Ile 325 330 335Ala Thr Ile Ser Pro Gly Ile Ser Ser Cys Glu Tyr Thr Leu Asn Thr 340 345350 Leu Arg Tyr Ala Asp Arg Val Lys Glu Leu Ser Pro His Ser Gly Pro 355360 365 Ser Gly Glu Gln Leu Ile Gln Met Glu Thr Glu Glu Met Glu Ala Cys370 375 380 Ser Asn Gly Ala Leu Ile Pro 385 390 5 1088 DNA Human 5atgacagaat atgtgtctgt gttaggaaac gcccactgaa taagcaagaa ttggccaaga 60aagaaattga tgtgatttcc attcctagca agtgtctcct cttggtacat gaacccaagt 120tgaaagtgga cttaacaaag tatctggaga accaagcatt ctgctttgac tttgcatttg 180atgaaacagc ttcgaatgaa gttgtctaca ggttcacagc aaggccactg gtacagacaa 240tctttgaagg tggaaaagca acttgttttg catatggcca gacaggaagt ggcaagacac 300atactatggg cggagacctc tctgggaaag cccagaatgc atccaaaggg atctatgcca 360tggcctcccg ggacgtcttc ctcctgaaga atcaaccctg ctaccggaag ttgggcctgg 420aagtctatgt gacattcttc gagatctaca atgggaagct gtttgacctg ctcaacaaga 480aggccaagct gcgcgtgctg gaggatggca agcaacaggt gcaagtggtg gggctgcagg 540agcatctggt taactctgct gatgatgtca tcaagatgat cgacatgggc agcgcctgca 600gaacctctgg gcagacattt gccaactcca attcctcccg ctcccacgcg tgcttccaaa 660ttattcttcg agctaaaggg agaatgcatg gcaagttctc tttggtagat ctggcaggga 720atgagcgagg cgcggacact tccagtgctg accggcagac ccgcatggag ggcgcagaaa 780tcaacaagag tctcttagcc ctgaaggagt gcatcagggc cctgggacag aacaaggctc 840acaccccgtt ccgtgagagc aagctgacac aggtgctgag ggactccttc attggggaga 900actctaggac ttgcatgatt gccacgatct caccaggcat aagctcctgt gaatatactt 960taaacaccct gagatatgca gacagggtca aggagctgag cccccacagt gggcccagtg 1020gagagcagtt gattcaaatg gaaacagaag agatggaagc ctgctctaac ggggcgctga 1080ttccatga 1088 6 362 PRT Human 6 Met His Arg Ile Cys Val Cys Val Arg LysArg Pro Leu Asn Lys Gln 1 5 10 15 Glu Leu Ala Lys Lys Glu Ile Asp ValIle Ser Ile Pro Ser Lys Cys 20 25 30 Leu Leu Leu Val His Glu Pro Lys LeuLys Val Asp Leu Thr Lys Tyr 35 40 45 Leu Glu Asn Gln Ala Phe Cys Phe AspPhe Ala Phe Asp Glu Thr Ala 50 55 60 Ser Asn Glu Val Val Tyr Arg Phe ThrAla Arg Pro Leu Val Gln Thr 65 70 75 80 Ile Phe Glu Gly Gly Lys Ala ThrCys Phe Ala Tyr Gly Gln Thr Gly 85 90 95 Ser Gly Lys Thr His Thr Met GlyGly Asp Leu Ser Gly Lys Ala Gln 100 105 110 Asn Ala Ser Lys Gly Ile TyrAla Met Ala Ser Arg Asp Val Phe Leu 115 120 125 Leu Lys Asn Gln Pro CysTyr Arg Lys Leu Gly Leu Glu Val Tyr Val 130 135 140 Thr Phe Phe Glu IleTyr Asn Gly Lys Leu Phe Asp Leu Leu Asn Lys 145 150 155 160 Lys Ala LysLeu Arg Val Leu Glu Asp Gly Lys Gln Gln Val Gln Val 165 170 175 Val GlyLeu Gln Glu His Leu Val Asn Ser Ala Asp Asp Val Ile Lys 180 185 190 MetIle Asp Met Gly Ser Ala Cys Arg Thr Ser Gly Gln Thr Phe Ala 195 200 205Asn Ser Asn Ser Ser Arg Ser His Ala Cys Phe Gln Ile Ile Leu Arg 210 215220 Ala Lys Gly Arg Met His Gly Lys Phe Ser Leu Val Asp Leu Ala Gly 225230 235 240 Asn Glu Arg Gly Ala Asp Thr Ser Ser Ala Asp Arg Gln Thr ArgMet 245 250 255 Glu Gly Ala Glu Ile Asn Lys Ser Leu Leu Ala Leu Lys GluCys Ile 260 265 270 Arg Ala Leu Gly Gln Asn Lys Ala His Thr Pro Phe ArgGlu Ser Lys 275 280 285 Leu Thr Gln Val Leu Arg Asp Ser Phe Ile Gly GluAsn Ser Arg Thr 290 295 300 Cys Met Ile Ala Thr Ile Ser Pro Gly Ile SerSer Cys Glu Tyr Thr 305 310 315 320 Leu Asn Thr Leu Arg Tyr Ala Asp ArgVal Lys Glu Leu Ser Pro His 325 330 335 Ser Gly Pro Ser Gly Glu Gln LeuIle Gln Met Glu Thr Glu Glu Met 340 345 350 Glu Ala Cys Ser Asn Gly AlaLeu Ile Pro 355 360 7 1420 DNA Human 7 atggaggaaa tcatgtcttg tgaaggaagtggaaaaaatg aagaacaagc gagaagagaa 60 gaaggcccag aactctgaaa tgagaatgaagagagctcag gagtatgaca gtagttttcc 120 aaactgggaa tttgcccgaa tgattaaagaatttcgggct actttggaat gtcatccact 180 tactatgact gatcctatcg aagagcacagaatatgtgtc tgtgttagga aacgcccact 240 gaataagcaa gaattggcca agaaagaaattgatgtgatt tccattccta gcaagtgtct 300 cctcttggta catgaaccca agttgaaagtggacttaaca aagtatctgg agaaccaagc 360 attctgcttt gactttgcat ttgatgaaacagcttcgaat gaagttgtct acaggttcac 420 agcaaggcca ctggtacaga caatctttgaaggtggaaaa gcaacttgtt ttgcatatgg 480 ccagacagga agtggcaaga cacatactatgggcggagac ctctctggga aagcccagaa 540 tgcatccaaa gggatctatg ccatggcctcccgggacgtc ttcctcctga agaatcaacc 600 ctgctaccgg aagttgggcc tggaagtctatgtgacattc ttcgagatct acaatgggaa 660 gctgtttgac ctgctcaaca agaaggccaagctgcgcgtg ctggaggatg gcaagcaaca 720 ggtgcaagtg gtggggctgc aggagcatctggttaactct gctgatgatg tcatcaagat 780 gatcgacatg ggcagcgcct gcagaacctctgggcagaca tttgccaact ccaattcctc 840 ccgctcccac gcgtgcttcc aaattattcttcgagctaaa gggagaatgc atggcaagtt 900 ctctttggta gatctggcag ggaatgagcgaggcgcggac acttccagtg ctgaccggca 960 gacccgcatg gagggcgcag aaatcaacaagagtctctta gccctgaagg agtgcatcag 1020 ggccctggga cagaacaagg ctcacaccccgttccgtgag agcaagctga cacaggtgct 1080 gagggactcc ttcattgggg agaactctaggacttgcatg attgccacga tctcaccagg 1140 cataagctcc tgtgaatata ctttaaacaccctgagatat gcagacaggg tcaaggagct 1200 gagcccccac agtgggccca gtggagagcagttgattcaa atggaaacag aagagatgga 1260 agcctgctct aacggggcgc tgattccaggcaatttatcc aaggaagagg aggaactgtc 1320 ttcccagatg tccagcttta acgaagccatgactcagatc agggagctgg aggagaaggc 1380 tatggaagag ctcaaggaga tcatacagcaaggaccatga 1420 8 473 PRT Human 8 Met Arg Arg Lys Ser Cys Leu Val LysGlu Val Glu Lys Met Lys Asn 1 5 10 15 Lys Arg Glu Glu Lys Lys Ala GlnAsn Ser Glu Met Arg Met Lys Arg 20 25 30 Ala Gln Glu Tyr Asp Ser Ser PhePro Asn Trp Glu Phe Ala Arg Met 35 40 45 Ile Lys Glu Phe Arg Ala Thr LeuGlu Cys His Pro Leu Thr Met Thr 50 55 60 Asp Pro Ile Glu Glu His Arg IleCys Val Cys Val Arg Lys Arg Pro 65 70 75 80 Leu Asn Lys Gln Glu Leu AlaLys Lys Glu Ile Asp Val Ile Ser Ile 85 90 95 Pro Ser Lys Cys Leu Leu LeuVal His Glu Pro Lys Leu Lys Val Asp 100 105 110 Leu Thr Lys Tyr Leu GluAsn Gln Ala Phe Cys Phe Asp Phe Ala Phe 115 120 125 Asp Glu Thr Ala SerAsn Glu Val Val Tyr Arg Phe Thr Ala Arg Pro 130 135 140 Leu Val Gln ThrIle Phe Glu Gly Gly Lys Ala Thr Cys Phe Ala Tyr 145 150 155 160 Gly GlnThr Gly Ser Gly Lys Thr His Thr Met Gly Gly Asp Leu Ser 165 170 175 GlyLys Ala Gln Asn Ala Ser Lys Gly Ile Tyr Ala Met Ala Ser Arg 180 185 190Asp Val Phe Leu Leu Lys Asn Gln Pro Cys Tyr Arg Lys Leu Gly Leu 195 200205 Glu Val Tyr Val Thr Phe Phe Glu Ile Tyr Asn Gly Lys Leu Phe Asp 210215 220 Leu Leu Asn Lys Lys Ala Lys Leu Arg Val Leu Glu Asp Gly Lys Gln225 230 235 240 Gln Val Gln Val Val Gly Leu Gln Glu His Leu Val Asn SerAla Asp 245 250 255 Asp Val Ile Lys Met Ile Asp Met Gly Ser Ala Cys ArgThr Ser Gly 260 265 270 Gln Thr Phe Ala Asn Ser Asn Ser Ser Arg Ser HisAla Cys Phe Gln 275 280 285 Ile Ile Leu Arg Ala Lys Gly Arg Met His GlyLys Phe Ser Leu Val 290 295 300 Asp Leu Ala Gly Asn Glu Arg Gly Ala AspThr Ser Ser Ala Asp Arg 305 310 315 320 Gln Thr Arg Met Glu Gly Ala GluIle Asn Lys Ser Leu Leu Ala Leu 325 330 335 Lys Glu Cys Ile Arg Ala LeuGly Gln Asn Lys Ala His Thr Pro Phe 340 345 350 Arg Glu Ser Lys Leu ThrGln Val Leu Arg Asp Ser Phe Ile Gly Glu 355 360 365 Asn Ser Arg Thr CysMet Ile Ala Thr Ile Ser Pro Gly Ile Ser Ser 370 375 380 Cys Glu Tyr ThrLeu Asn Thr Leu Arg Tyr Ala Asp Arg Val Lys Glu 385 390 395 400 Leu SerPro His Ser Gly Pro Ser Gly Glu Gln Leu Ile Gln Met Glu 405 410 415 ThrGlu Glu Met Glu Ala Cys Ser Asn Gly Ala Leu Ile Pro Gly Asn 420 425 430Leu Ser Lys Glu Glu Glu Glu Leu Ser Ser Gln Met Ser Ser Phe Asn 435 440445 Glu Ala Met Thr Gln Ile Arg Glu Leu Glu Glu Lys Ala Met Glu Glu 450455 460 Leu Lys Glu Ile Ile Gln Gln Gly Pro 465 470 9 1304 DNA Human 9atgcaaactg ggaatttgcc cgaatgatta aagaatttcg ggctactttg gaatgtcatc 60cacttactat gactgatcct atcgaagagc acagaatatg tgtctgtgtt aggaaacgcc 120cactgaataa gcaagaattg gccaagaaag aaattgatgt gatttccatt cctagcaagt 180gtctcctctt ggtacatgaa cccaagttga aagtggactt aacaaagtat ctggagaacc 240aagcattctg ctttgacttt gcatttgatg aaacagcttc gaatgaagtt gtctacaggt 300tcacagcaag gccactggta cagacaatct ttgaaggtgg aaaagcaact tgttttgcat 360atggccagac aggaagtggc aagacacata ctatgggcgg agacctctct gggaaagccc 420agaatgcatc caaagggatc tatgccatgg cctcccggga cgtcttcctc ctgaagaatc 480aaccctgcta ccggaagttg ggcctggaag tctatgtgac attcttcgag atctacaatg 540ggaagctgtt tgacctgctc aacaagaagg ccaagctgcg cgtgctggag gatggcaagc 600aacaggtgca agtggtgggg ctgcaggagc atctggttaa ctctgctgat gatgtcatca 660agatgatcga catgggcagc gcctgcagaa cctctgggca gacatttgcc aactccaatt 720cctcccgctc ccacgcgtgc ttccaaatta ttcttcgagc taaagggaga atgcatggca 780agttctcttt ggtagatctg gcagggaatg agcgaggcgc ggacacttcc agtgctgacc 840ggcagacccg catggagggc gcagaaatca acaagagtct cttagccctg aaggagtgca 900tcagggccct gggacagaac aaggctcaca ccccgttccg tgagagcaag ctgacacagg 960tgctgaggga ctccttcatt ggggagaact ctaggacttg catgattgcc acgatctcac 1020caggcataag ctcctgtgaa tatactttaa acaccctgag atatgcagac agggtcaagg 1080agctgagccc ccacagtggg cccagtggag agcagttgat tcaaatggaa acagaagaga 1140tggaagcctg ctctaacggg gcgctgattc caggcaattt atccaaggaa gaggaggaac 1200tgtcttccca gatgtccagc tttaacgaag ccatgactca gatcagggag ctggaggaga 1260aggctatgga agagctcaag gagatcatac agcaaggacc atga 1304 10 434 PRT Human10 Met Pro Asn Trp Glu Phe Ala Arg Met Ile Lys Glu Phe Arg Ala Thr 1 510 15 Leu Glu Cys His Pro Leu Thr Met Thr Asp Pro Ile Glu Glu His Arg 2025 30 Ile Cys Val Cys Val Arg Lys Arg Pro Leu Asn Lys Gln Glu Leu Ala 3540 45 Lys Lys Glu Ile Asp Val Ile Ser Ile Pro Ser Lys Cys Leu Leu Leu 5055 60 Val His Glu Pro Lys Leu Lys Val Asp Leu Thr Lys Tyr Leu Glu Asn 6570 75 80 Gln Ala Phe Cys Phe Asp Phe Ala Phe Asp Glu Thr Ala Ser Asn Glu85 90 95 Val Val Tyr Arg Phe Thr Ala Arg Pro Leu Val Gln Thr Ile Phe Glu100 105 110 Gly Gly Lys Ala Thr Cys Phe Ala Tyr Gly Gln Thr Gly Ser GlyLys 115 120 125 Thr His Thr Met Gly Gly Asp Leu Ser Gly Lys Ala Gln AsnAla Ser 130 135 140 Lys Gly Ile Tyr Ala Met Ala Ser Arg Asp Val Phe LeuLeu Lys Asn 145 150 155 160 Gln Pro Cys Tyr Arg Lys Leu Gly Leu Glu ValTyr Val Thr Phe Phe 165 170 175 Glu Ile Tyr Asn Gly Lys Leu Phe Asp LeuLeu Asn Lys Lys Ala Lys 180 185 190 Leu Arg Val Leu Glu Asp Gly Lys GlnGln Val Gln Val Val Gly Leu 195 200 205 Gln Glu His Leu Val Asn Ser AlaAsp Asp Val Ile Lys Met Ile Asp 210 215 220 Met Gly Ser Ala Cys Arg ThrSer Gly Gln Thr Phe Ala Asn Ser Asn 225 230 235 240 Ser Ser Arg Ser HisAla Cys Phe Gln Ile Ile Leu Arg Ala Lys Gly 245 250 255 Arg Met His GlyLys Phe Ser Leu Val Asp Leu Ala Gly Asn Glu Arg 260 265 270 Gly Ala AspThr Ser Ser Ala Asp Arg Gln Thr Arg Met Glu Gly Ala 275 280 285 Glu IleAsn Lys Ser Leu Leu Ala Leu Lys Glu Cys Ile Arg Ala Leu 290 295 300 GlyGln Asn Lys Ala His Thr Pro Phe Arg Glu Ser Lys Leu Thr Gln 305 310 315320 Val Leu Arg Asp Ser Phe Ile Gly Glu Asn Ser Arg Thr Cys Met Ile 325330 335 Ala Thr Ile Ser Pro Gly Ile Ser Ser Cys Glu Tyr Thr Leu Asn Thr340 345 350 Leu Arg Tyr Ala Asp Arg Val Lys Glu Leu Ser Pro His Ser GlyPro 355 360 365 Ser Gly Glu Gln Leu Ile Gln Met Glu Thr Glu Glu Met GluAla Cys 370 375 380 Ser Asn Gly Ala Leu Ile Pro Gly Asn Leu Ser Lys GluGlu Glu Glu 385 390 395 400 Leu Ser Ser Gln Met Ser Ser Phe Asn Glu AlaMet Thr Gln Ile Arg 405 410 415 Glu Leu Glu Glu Lys Ala Met Glu Glu LeuLys Glu Ile Ile Gln Gln 420 425 430 Gly Pro 11 1217 DNA Human 11atgacagaat atgtgtctgt gttaggaaac gcccactgaa taagcaagaa ttggccaaga 60aagaaattga tgtgatttcc attcctagca agtgtctcct cttggtacat gaacccaagt 120tgaaagtgga cttaacaaag tatctggaga accaagcatt ctgctttgac tttgcatttg 180atgaaacagc ttcgaatgaa gttgtctaca ggttcacagc aaggccactg gtacagacaa 240tctttgaagg tggaaaagca acttgttttg catatggcca gacaggaagt ggcaagacac 300atactatggg cggagacctc tctgggaaag cccagaatgc atccaaaggg atctatgcca 360tggcctcccg ggacgtcttc ctcctgaaga atcaaccctg ctaccggaag ttgggcctgg 420aagtctatgt gacattcttc gagatctaca atgggaagct gtttgacctg ctcaacaaga 480aggccaagct gcgcgtgctg gaggatggca agcaacaggt gcaagtggtg gggctgcagg 540agcatctggt taactctgct gatgatgtca tcaagatgat cgacatgggc agcgcctgca 600gaacctctgg gcagacattt gccaactcca attcctcccg ctcccacgcg tgcttccaaa 660ttattcttcg agctaaaggg agaatgcatg gcaagttctc tttggtagat ctggcaggga 720atgagcgagg cgcggacact tccagtgctg accggcagac ccgcatggag ggcgcagaaa 780tcaacaagag tctcttagcc ctgaaggagt gcatcagggc cctgggacag aacaaggctc 840acaccccgtt ccgtgagagc aagctgacac aggtgctgag ggactccttc attggggaga 900actctaggac ttgcatgatt gccacgatct caccaggcat aagctcctgt gaatatactt 960taaacaccct gagatatgca gacagggtca aggagctgag cccccacagt gggcccagtg 1020gagagcagtt gattcaaatg gaaacagaag agatggaagc ctgctctaac ggggcgctga 1080ttccaggcaa tttatccaag gaagaggagg aactgtcttc ccagatgtcc agctttaacg 1140aagccatgac tcagatcagg gagctggagg agaaggctat ggaagagctc aaggagatca 1200tacagcaagg accatga 1217 12 405 PRT Human 12 Met His Arg Ile Cys Val CysVal Arg Lys Arg Pro Leu Asn Lys Gln 1 5 10 15 Glu Leu Ala Lys Lys GluIle Asp Val Ile Ser Ile Pro Ser Lys Cys 20 25 30 Leu Leu Leu Val His GluPro Lys Leu Lys Val Asp Leu Thr Lys Tyr 35 40 45 Leu Glu Asn Gln Ala PheCys Phe Asp Phe Ala Phe Asp Glu Thr Ala 50 55 60 Ser Asn Glu Val Val TyrArg Phe Thr Ala Arg Pro Leu Val Gln Thr 65 70 75 80 Ile Phe Glu Gly GlyLys Ala Thr Cys Phe Ala Tyr Gly Gln Thr Gly 85 90 95 Ser Gly Lys Thr HisThr Met Gly Gly Asp Leu Ser Gly Lys Ala Gln 100 105 110 Asn Ala Ser LysGly Ile Tyr Ala Met Ala Ser Arg Asp Val Phe Leu 115 120 125 Leu Lys AsnGln Pro Cys Tyr Arg Lys Leu Gly Leu Glu Val Tyr Val 130 135 140 Thr PhePhe Glu Ile Tyr Asn Gly Lys Leu Phe Asp Leu Leu Asn Lys 145 150 155 160Lys Ala Lys Leu Arg Val Leu Glu Asp Gly Lys Gln Gln Val Gln Val 165 170175 Val Gly Leu Gln Glu His Leu Val Asn Ser Ala Asp Asp Val Ile Lys 180185 190 Met Ile Asp Met Gly Ser Ala Cys Arg Thr Ser Gly Gln Thr Phe Ala195 200 205 Asn Ser Asn Ser Ser Arg Ser His Ala Cys Phe Gln Ile Ile LeuArg 210 215 220 Ala Lys Gly Arg Met His Gly Lys Phe Ser Leu Val Asp LeuAla Gly 225 230 235 240 Asn Glu Arg Gly Ala Asp Thr Ser Ser Ala Asp ArgGln Thr Arg Met 245 250 255 Glu Gly Ala Glu Ile Asn Lys Ser Leu Leu AlaLeu Lys Glu Cys Ile 260 265 270 Arg Ala Leu Gly Gln Asn Lys Ala His ThrPro Phe Arg Glu Ser Lys 275 280 285 Leu Thr Gln Val Leu Arg Asp Ser PheIle Gly Glu Asn Ser Arg Thr 290 295 300 Cys Met Ile Ala Thr Ile Ser ProGly Ile Ser Ser Cys Glu Tyr Thr 305 310 315 320 Leu Asn Thr Leu Arg TyrAla Asp Arg Val Lys Glu Leu Ser Pro His 325 330 335 Ser Gly Pro Ser GlyGlu Gln Leu Ile Gln Met Glu Thr Glu Glu Met 340 345 350 Glu Ala Cys SerAsn Gly Ala Leu Ile Pro Gly Asn Leu Ser Lys Glu 355 360 365 Glu Glu GluLeu Ser Ser Gln Met Ser Ser Phe Asn Glu Ala Met Thr 370 375 380 Gln IleArg Glu Leu Glu Glu Lys Ala Met Glu Glu Leu Lys Glu Ile 385 390 395 400Ile Gln Gln Gly Pro 405 13 2172 DNA Human 13 atggactcgt cgcttcaggcccgcctgttt cccggtctcg ctatcaagat ccaacgcagt 60 aatggtttaa ttcacagtgccaatgtaagg actgtgaact tggagaaatc ctgtgtttca 120 gtggaatggg cagaaggaggtgccacaaag ggcaaagaga ttgattttga tgatgtggct 180 gcaataaacc cagaactcttacagcttctt cccttacatc cgaaggacaa tctgcccttg 240 caggaaaatg taacaatccagaaacaaaaa cggagatccg tcaactccaa aattcctgct 300 ccaaaagaaa gtcttcgaagccgctccact cgcatgtcca ctgtctcaga gcttcgcatc 360 acggctcagg agaatgacatggaggtggag ctgcctgcag ctgcaaactc ccgcaagcag 420 ttttcagttc ctcctgcccccactaggcct tcctgccctg cagtggctga aataccattg 480 aggatggtca gcgaggagatggaagagcaa gtccattcca tccgtggcag ctcttctgca 540 aaccctgtga actcagttcggaggaaatca tgtcttgtga aggaagtgga aaaaatgaag 600 aacaagcgag aagagaagaaggcccagaac tctgaaatga gaatgaagag agctcaggag 660 tatgacagta gttttccaaactgggaattt gcccgaatga ttaaagaatt tcgggctact 720 ttggaatgtc atccacttactatgactgat cctatcgaag agcacagaat atgtgtctgt 780 gttaggaaac gcccactgaataagcaagaa ttggccaaga aagaaattga tgtgatttcc 840 attcctagca agtgtctcctcttggtacat gaacccaagt tgaaagtgga cttaacaaag 900 tatctggaga accaagcattctgctttgac tttgcatttg atgaaacagc ttcgaatgaa 960 gttgtctaca ggttcacagcaaggccactg gtacagacaa tctttgaagg tggaaaagca 1020 acttgttttg catatggccagacaggaagt ggcaagacac atactatggg cggagacctc 1080 tctgggaaag cccagaatgcatccaaaggg atctatgcca tggcctcccg ggacgtcttc 1140 ctcctgaaga atcaaccctgctaccggaag ttgggcctgg aagtctatgt gacattcttc 1200 gagatctaca atgggaagctgtttgacctg ctcaacaaga aggccaagct gcgcgtgctg 1260 gaggatggca agcaacaggtgcaagtggtg gggctgcagg agcatctggt taactctgct 1320 gatgatgtca tcaagatgatcgacatgggc agcgcctgca gaacctctgg gcagacattt 1380 gccaactcca attcctcccgctcccacgcg tgcttccaaa ttattcttcg agctaaaggg 1440 agaatgcatg gcaagttctctttggtagat ctggcaggga atgagcgagg cgcggacact 1500 tccagtgctg accggcagacccgcatggag ggcgcagaaa tcaacaagag tctcttagcc 1560 ctgaaggagt gcatcagggccctgggacag aacaaggctc acaccccgtt ccgtgagagc 1620 aagctgacac aggtgctgagggactccttc attggggaga actctaggac ttgcatgatt 1680 gccacgatct caccaggcataagctcctgt gaatatactt taaacaccct gagatatgca 1740 gacagggtca aggagctgagcccccacagt gggcccagtg gagagcagtt gattcaaatg 1800 gaaacagaag agatggaagcctgctctaac ggggcgctga ttccaggcaa tttatccaag 1860 gaagaggagg aactgtcttcccagatgtcc agctttaacg aagccatgac tcagatcagg 1920 gagctggagg agaaggctatggaagagctc aaggagatca tacagcaagg accagactgg 1980 cttgagctct ctgagatgaccgagcagcca gactatgacc tggagacctt tgtgaacaaa 2040 gcggaatctg ctctggcccagcaagccaag catttctcag ccctgcgaga tgtcatcaag 2100 gccttacgcc tggccatgcagctggaagag caggctagca gacaaataag cagcaagaaa 2160 cggccccagt ga 2172 14723 PRT Human 14 Met Asp Ser Ser Leu Gln Ala Arg Leu Phe Pro Gly Leu AlaIle Lys 1 5 10 15 Ile Gln Arg Ser Asn Gly Leu Ile His Ser Ala Asn ValArg Thr Val 20 25 30 Asn Leu Glu Lys Ser Cys Val Ser Val Glu Trp Ala GluGly Gly Ala 35 40 45 Thr Lys Gly Lys Glu Ile Asp Phe Asp Asp Val Ala AlaIle Asn Pro 50 55 60 Glu Leu Leu Gln Leu Leu Pro Leu His Pro Lys Asp AsnLeu Pro Leu 65 70 75 80 Gln Glu Asn Val Thr Ile Gln Lys Gln Lys Arg ArgSer Val Asn Ser 85 90 95 Lys Ile Pro Ala Pro Lys Glu Ser Leu Arg Ser ArgSer Thr Arg Met 100 105 110 Ser Thr Val Ser Glu Leu Arg Ile Thr Ala GlnGlu Asn Asp Met Glu 115 120 125 Val Glu Leu Pro Ala Ala Ala Asn Ser ArgLys Gln Phe Ser Val Pro 130 135 140 Pro Ala Pro Thr Arg Pro Ser Cys ProAla Val Ala Glu Ile Pro Leu 145 150 155 160 Arg Met Val Ser Glu Glu MetGlu Glu Gln Val His Ser Ile Arg Gly 165 170 175 Ser Ser Ser Ala Asn ProVal Asn Ser Val Arg Arg Lys Ser Cys Leu 180 185 190 Val Lys Glu Val GluLys Met Lys Asn Lys Arg Glu Glu Lys Lys Ala 195 200 205 Gln Asn Ser GluMet Arg Met Lys Arg Ala Gln Glu Tyr Asp Ser Ser 210 215 220 Phe Pro AsnTrp Glu Phe Ala Arg Met Ile Lys Glu Phe Arg Ala Thr 225 230 235 240 LeuGlu Cys His Pro Leu Thr Met Thr Asp Pro Ile Glu Glu His Arg 245 250 255Ile Cys Val Cys Val Arg Lys Arg Pro Leu Asn Lys Gln Glu Leu Ala 260 265270 Lys Lys Glu Ile Asp Val Ile Ser Ile Pro Ser Lys Cys Leu Leu Leu 275280 285 Val His Glu Pro Lys Leu Lys Val Asp Leu Thr Lys Tyr Leu Glu Asn290 295 300 Gln Ala Phe Cys Phe Asp Phe Ala Phe Asp Glu Thr Ala Ser AsnGlu 305 310 315 320 Val Val Tyr Arg Phe Thr Ala Arg Pro Leu Val Gln ThrIle Phe Glu 325 330 335 Gly Gly Lys Ala Thr Cys Phe Ala Tyr Gly Gln ThrGly Ser Gly Lys 340 345 350 Thr His Thr Met Gly Gly Asp Leu Ser Gly LysAla Gln Asn Ala Ser 355 360 365 Lys Gly Ile Tyr Ala Met Ala Ser Arg AspVal Phe Leu Leu Lys Asn 370 375 380 Gln Pro Cys Tyr Arg Lys Leu Gly LeuGlu Val Tyr Val Thr Phe Phe 385 390 395 400 Glu Ile Tyr Asn Gly Lys LeuPhe Asp Leu Leu Asn Lys Lys Ala Lys 405 410 415 Leu Arg Val Leu Glu AspGly Lys Gln Gln Val Gln Val Val Gly Leu 420 425 430 Gln Glu His Leu ValAsn Ser Ala Asp Asp Val Ile Lys Met Ile Asp 435 440 445 Met Gly Ser AlaCys Arg Thr Ser Gly Gln Thr Phe Ala Asn Ser Asn 450 455 460 Ser Ser ArgSer His Ala Cys Phe Gln Ile Ile Leu Arg Ala Lys Gly 465 470 475 480 ArgMet His Gly Lys Phe Ser Leu Val Asp Leu Ala Gly Asn Glu Arg 485 490 495Gly Ala Asp Thr Ser Ser Ala Asp Arg Gln Thr Arg Met Glu Gly Ala 500 505510 Glu Ile Asn Lys Ser Leu Leu Ala Leu Lys Glu Cys Ile Arg Ala Leu 515520 525 Gly Gln Asn Lys Ala His Thr Pro Phe Arg Glu Ser Lys Leu Thr Gln530 535 540 Val Leu Arg Asp Ser Phe Ile Gly Glu Asn Ser Arg Thr Cys MetIle 545 550 555 560 Ala Thr Ile Ser Pro Gly Ile Ser Ser Cys Glu Tyr ThrLeu Asn Thr 565 570 575 Leu Arg Tyr Ala Asp Arg Val Lys Glu Leu Ser ProHis Ser Gly Pro 580 585 590 Ser Gly Glu Gln Leu Ile Gln Met Glu Thr GluGlu Met Glu Ala Cys 595 600 605 Ser Asn Gly Ala Leu Ile Pro Gly Asn LeuSer Lys Glu Glu Glu Glu 610 615 620 Leu Ser Ser Gln Met Ser Ser Phe AsnGlu Ala Met Thr Gln Ile Arg 625 630 635 640 Glu Leu Glu Glu Lys Ala MetGlu Glu Leu Lys Glu Ile Ile Gln Gln 645 650 655 Gly Pro Asp Trp Leu GluLeu Ser Glu Met Thr Glu Gln Pro Asp Tyr 660 665 670 Asp Leu Glu Thr PheVal Asn Lys Ala Glu Ser Ala Leu Ala Gln Gln 675 680 685 Ala Lys His PheSer Ala Leu Arg Asp Val Ile Lys Ala Leu Arg Leu 690 695 700 Ala Met GlnLeu Glu Glu Gln Ala Ser Arg Gln Ile Ser Ser Lys Lys 705 710 715 720 ArgPro Gln 15 2740 DNA Human 15 gcgaaattga ggtttcttgg tattgcgcgt ttctcttccttgctgactct ccgaatggcc 60 atggactcgt cgcttcaggc ccgcctgttt cccggtctcgctatcaagat ccaacgcagt 120 aatggtttaa ttcacagtgc caatgtaagg actgtgaacttggagaaatc ctgtgtttca 180 gtggaatggg cagaaggagg tgccacaaag ggcaaagagattgattttga tgatgtggct 240 gcaataaacc cagaactctt acagcttctt cccttacatccgaaggacaa tctgcccttg 300 caggaaaatg taacaatcca gaaacaaaaa cggagatccgtcaactccaa aattcctgct 360 ccaaaagaaa gtcttcgaag ccgctccact cgcatgtccactgtctcaga gcttcgcatc 420 acggctcagg agaatgacat ggaggtggag ctgcctgcagctgcaaactc ccgcaagcag 480 ttttcagttc ctcctgcccc cactaggcct tcctgccctgcagtggctga aataccattg 540 aggatggtca gcgaggagat ggaagagcaa gtccattccatccgtggcag ctcttctgca 600 aaccctgtga actcagttcg gaggaaatca tgtcttgtgaaggaagtgga aaaaatgaag 660 aacaagcgag aagagaagaa ggcccagaac tctgaaatgagaatgaagag agctcaggag 720 tatgacagta gttttccaaa ctgggaattt gcccgaatgattaaagaatt tcgggctact 780 ttggaatgtc atccacttac tatgactgat cctatcgaagagcacagaat atgtgtctgt 840 gttaggaaac gcccactgaa taagcaagaa ttggccaagaaagaaattga tgtgatttcc 900 attcctagca agtgtctcct cttggtacat gaacccaagttgaaagtgga cttaacaaag 960 tatctggaga accaagcatt ctgctttgac tttgcatttgatgaaacagc ttcgaatgaa 1020 gttgtctaca ggttcacagc aaggccactg gtacagacaatctttgaagg tggaaaagca 1080 acttgttttg catatggcca gacaggaagt ggcaagacacatactatggg cggagacctc 1140 tctgggaaag cccagaatgc atccaaaggg atctatgccatggcctcccg ggacgtcttc 1200 ctcctgaaga atcaaccctg ctaccggaag ttgggcctggaagtctatgt gacattcttc 1260 gagatctaca atgggaagct gtttgacctg ctcaacaagaaggccaagct gcgcgtgctg 1320 gaggacggca agcaacaggt gcaagtggtg gggctgcaggagcatctggt taactctgct 1380 gatgatgtca tcaagatgct cgacatgggc agcgcctgcagaacctctgg gcagacattt 1440 gccaactcca attcctcccg ctcccacgcg tgcttccaaattattcttcg agctaaaggg 1500 agaatgcatg gcaagttctc tttggtagat ctggcagggaatgagcgagg cgcagacact 1560 tccagtgctg accggcagac ccgcatggag ggcgcagaaatcaacaagag tctcttagcc 1620 ctgaaggagt gcatcagggc cctgggacag aacaaggctcacaccccgtt ccgtgagagc 1680 aagctgacac aggtgctgag ggactccttc attggggagaactctaggac ttgcatgatt 1740 gccacgatct caccaggcat aagctcctgt gaatatactttaaacaccct gagatatgca 1800 gacagggtca aggagctgag cccccacagt gggcccagtggagagcagtt gattcaaatg 1860 gaaacagaag agatggaagc ctgctctaac ggggcgctgattccaggcaa tttatccaag 1920 gaagaggagg aactgtcttc ccagatgtcc agctttaacgaagccatgac tcagatcagg 1980 gagctggagg agaaggctat ggaagagctc aaggagatcatacagcaagg accagactgg 2040 cttgagctct ctgagatgac cgagcagcca gactatgacctggagacctt tgtgaacaaa 2100 gcggaatctg ctctggccca gcaagccaag catttctcagccctgcgaga tgtcatcaag 2160 gccttacgcc tggccatgca gctggaagag caggctagcagacaaataag cagcaagaaa 2220 cggccccagt gacgactgca aataaaaatc tgtttggtttgacacccagc ctcttccctg 2280 gccctcccca gagaactttg ggtacctggt gggtctaggcagggtctgag ctgggacagg 2340 ttctggtaaa tgccaagtat gggggcatct gggcccagggcagctgggga gggggtcaga 2400 gtgacatggg acactccttt tctgttcctc agttgtcgccctcacgagag gaaggagctc 2460 ttagttaccc ttttgtgttg cccttctttc catcaaggggaatgttctca gcatagagct 2520 ttctccgcag catcctgcct gcgtggactg gctgctaatggagagctccc tggggttgtc 2580 ctggctctgg ggagagagac ggagccttta gtacagctatctgctggctc taaaccttct 2640 acgcctttgg gccgagcact gaatgtcttg tactttaaaaaaatgtttct gagacctctt 2700 tctactttac tgtctcccta gagtcctaga ggatccctac2740 16 725 PRT Human 16 Met Ala Met Asp Ser Ser Leu Gln Ala Arg Leu PhePro Gly Leu Ala 1 5 10 15 Ile Lys Ile Gln Arg Ser Asn Gly Leu Ile HisSer Ala Asn Val Arg 20 25 30 Thr Val Asn Leu Glu Lys Ser Cys Val Ser ValGlu Trp Ala Glu Gly 35 40 45 Gly Ala Thr Lys Gly Lys Glu Ile Asp Phe AspAsp Val Ala Ala Ile 50 55 60 Asn Pro Glu Leu Leu Gln Leu Leu Pro Leu HisPro Lys Asp Asn Leu 65 70 75 80 Pro Leu Gln Glu Asn Val Thr Ile Gln LysGln Lys Arg Arg Ser Val 85 90 95 Asn Ser Lys Ile Pro Ala Pro Lys Glu SerLeu Arg Ser Arg Ser Thr 100 105 110 Arg Met Ser Thr Val Ser Glu Leu ArgIle Thr Ala Gln Glu Asn Asp 115 120 125 Met Glu Val Glu Leu Pro Ala AlaAla Asn Ser Arg Lys Gln Phe Ser 130 135 140 Val Pro Pro Ala Pro Thr ArgPro Ser Cys Pro Ala Val Ala Glu Ile 145 150 155 160 Pro Leu Arg Met ValSer Glu Glu Met Glu Glu Gln Val His Ser Ile 165 170 175 Arg Gly Ser SerSer Ala Asn Pro Val Asn Ser Val Arg Arg Lys Ser 180 185 190 Cys Leu ValLys Glu Val Glu Lys Met Lys Asn Lys Arg Glu Glu Lys 195 200 205 Lys AlaGln Asn Ser Glu Met Arg Met Lys Arg Ala Gln Glu Tyr Asp 210 215 220 SerSer Phe Pro Asn Trp Glu Phe Ala Arg Met Ile Lys Glu Phe Arg 225 230 235240 Ala Thr Leu Glu Cys His Pro Leu Thr Met Thr Asp Pro Ile Glu Glu 245250 255 His Arg Ile Cys Val Cys Val Arg Lys Arg Pro Leu Asn Lys Gln Glu260 265 270 Leu Ala Lys Lys Glu Ile Asp Val Ile Ser Ile Pro Ser Lys CysLeu 275 280 285 Leu Leu Val His Glu Pro Lys Leu Lys Val Asp Leu Thr LysTyr Leu 290 295 300 Glu Asn Gln Ala Phe Cys Phe Asp Phe Ala Phe Asp GluThr Ala Ser 305 310 315 320 Asn Glu Val Val Tyr Arg Phe Thr Ala Arg ProLeu Val Gln Thr Ile 325 330 335 Phe Glu Gly Gly Lys Ala Thr Cys Phe AlaTyr Gly Gln Thr Gly Ser 340 345 350 Gly Lys Thr His Thr Met Gly Gly AspLeu Ser Gly Lys Ala Gln Asn 355 360 365 Ala Ser Lys Gly Ile Tyr Ala MetAla Ser Arg Asp Val Phe Leu Leu 370 375 380 Lys Asn Gln Pro Cys Tyr ArgLys Leu Gly Leu Glu Val Tyr Val Thr 385 390 395 400 Phe Phe Glu Ile TyrAsn Gly Lys Leu Phe Asp Leu Leu Asn Lys Lys 405 410 415 Ala Lys Leu ArgVal Leu Glu Asp Gly Lys Gln Gln Val Gln Val Val 420 425 430 Gly Leu GlnGlu His Leu Val Asn Ser Ala Asp Asp Val Ile Lys Met 435 440 445 Leu AspMet Gly Ser Ala Cys Arg Thr Ser Gly Gln Thr Phe Ala Asn 450 455 460 SerAsn Ser Ser Arg Ser His Ala Cys Phe Gln Ile Ile Leu Arg Ala 465 470 475480 Lys Gly Arg Met His Gly Lys Phe Ser Leu Val Asp Leu Ala Gly Asn 485490 495 Glu Arg Gly Ala Asp Thr Ser Ser Ala Asp Arg Gln Thr Arg Met Glu500 505 510 Gly Ala Glu Ile Asn Lys Ser Leu Leu Ala Leu Lys Glu Cys IleArg 515 520 525 Ala Leu Gly Gln Asn Lys Ala His Thr Pro Phe Arg Glu SerLys Leu 530 535 540 Thr Gln Val Leu Arg Asp Ser Phe Ile Gly Glu Asn SerArg Thr Cys 545 550 555 560 Met Ile Ala Thr Ile Ser Pro Gly Ile Ser SerCys Glu Tyr Thr Leu 565 570 575 Asn Thr Leu Arg Tyr Ala Asp Arg Val LysGlu Leu Ser Pro His Ser 580 585 590 Gly Pro Ser Gly Glu Gln Leu Ile GlnMet Glu Thr Glu Glu Met Glu 595 600 605 Ala Cys Ser Asn Gly Ala Leu IlePro Gly Asn Leu Ser Lys Glu Glu 610 615 620 Glu Glu Leu Ser Ser Gln MetSer Ser Phe Asn Glu Ala Met Thr Gln 625 630 635 640 Ile Arg Glu Leu GluGlu Lys Ala Met Glu Glu Leu Lys Glu Ile Ile 645 650 655 Gln Gln Gly ProAsp Trp Leu Glu Leu Ser Glu Met Thr Glu Gln Pro 660 665 670 Asp Tyr AspLeu Glu Thr Phe Val Asn Lys Ala Glu Ser Ala Leu Ala 675 680 685 Gln GlnAla Lys His Phe Ser Ala Leu Arg Asp Val Ile Lys Ala Leu 690 695 700 ArgLeu Ala Met Gln Leu Glu Glu Gln Ala Ser Arg Gln Ile Ser Ser 705 710 715720 Lys Lys Arg Pro Gln 725

What is claimed is:
 1. An isolated protein, wherein the protein has anamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
 2. The protein ofclaim 1, wherein the protein is tagged at its C-terminal with a mycepitope and six histidines.
 3. The protein of claim 1, wherein theprotein is tagged at its N-tenninus with a T7 epitope.
 4. The protein ofclaim 1, wherein the protein is tagged at its N-terminus with a T7epitope.
 5. The protein of claim 1, wherein the protein has an almo acidsequence of SEQ ID NO.2.
 6. The protein of claim 1, wherein the proteinhas an amino acid sequence of SEQ ID NO:4.
 7. The protein of claim 1,wherein the protein bas an amino acid sequence of SEQ ID NO:6.
 8. Theprotein of claim 1, wherein the protein has an amino acid sequence ofSEQ ID NO:8.
 9. The protein of claim 1, wherein the protein has an aminoacid sequence of SEQ ID NO:10.
 10. The protein of claim 1, wherein theprotein has an amino acid sequence of SEQ ID NO:12.
 11. The protein ofclaim 1, wherein the protein has an amino acid sequence of SEQ ID NO:14.